PMC:2724026 / 12555-16861 JSONTXT

{"project":"2_test","denotations":[{"id":"19362094-1569556-62520522","span":{"begin":480,"end":482},"obj":"1569556"},{"id":"19362094-18538343-62520523","span":{"begin":656,"end":658},"obj":"18538343"}],"text":"Φ-Value analysis\nΦ-Values obtained were either zero or fractional, with no Φ-value of 1 (Tables 2 and 3), indicating no part of the protein is as fully formed in the transition state as it is in the native state. To interpret a Φ-value analysis, it is customary to consider Φ-value patterns, rather than to try to interpret individual Φ-values. This allows one to determine which regions of the protein are fully unfolded, partially folded or fully folded in the transition state.28 The Φ-values obtained are thus generally classified into low, medium and high classes, with the boundaries chosen to reflect the overall Φ-value distribution (e.g. see Ref. 29): in our case, the cut-offs used are: low, Φ ≤ 0.15; medium, Φ = 0.16–0.5; high, Φ ≥ 0.51.\nWe will consider the structure of the transition state of FADD DD in terms of formation of the three structural cores, i.e. formation of the two three-helix bundles B1 and B2, and the formation of the central core (the four-helix motif). (A detailed analysis of each helix is given in the Supplementary Data). The Φ-values were mapped on the native structure, to allow the patterns of Φ-values to be interpreted.\n\nFormation of B1 (formed by H1, H5 and H6): (Fig. 3)\nFormation of the B1 core was probed by six mutations: H1, V103A; H5, V162A, L165A; and H6, V173A, L176A, V177A. The extent of helix formation was probed by six Ala to Gly mutations: H1, A98G, A102G; H5, A166G, A167G; and H6, A175G, A178G.\nAll three helices are partially folded but the B1 core can be considered as only very weakly structured. Only H5 and H6 make contact through the B1 core, through interactions between L165 at the C-terminus of H5 and V173 at the N-terminus of H6. H1 is only connected to the B1 core in the native state via V103, but the Φ-value of V103A is zero, suggesting that H1 does not contribute to the formation of the B1 core in the transition state (TS). (Note, however, that H1 and H5 do interact in the TS, through sidechain contacts on the central core side of the helices).\n\nFormation of B2 (formed by H2, H3 and H4): (Fig. 3)\nFormation of the B2 core was probed by six mutations: H2, W112A; H3, I126A, I129A, Y133A; and H4, R140A, S144A. The extent of helix formation was probed by 12 Ala to Gly mutations: H2, A113G, A114G, A117G; H3, A127G, A131G, A132G; and H4, A138G, A139G, A142G, A143G, A146G, A150G.\nHelix 4 is the most structured region of the entire protein, with high Ala to Gly Φ-values at the N-terminal end, becoming lower towards the C-terminal end. The two B2 core residues in this helix both have high Φ-values; W112 (the only hydrophobic residue from H2 which packs into this core) has a medium Φ-value. All the Φ-values of helix 3 are close to zero; this helix plays no role in the formation of B2 in the TS. Thus, we infer that the B2 core is loosely structured in the TS with H2 packing onto H4.\n\nFormation of central core formed by H1, H2, H4 and H5 (Fig. 3)\nFormation of the central core was probed by nine mutations: H1, F101A, I104A; H2, L115M, L119M; H4, V141A, L145M, W148F; and H5, H160A, L161A. The extent of helix formation was probed by 13 Ala to Gly mutations: H1, A98G, A102G; H2, A113G, A114G, A117G; H4, A138G, A139G, A142G, A143G, A146G, A150G; and H5, A166G, S167G. (Note that in native FADD DD, H1 and H5 of the central core run parallel with each other and are packed orthogonally onto the parallel helix pair H2 and H4; Fig. 1).\nIn the TS, the central core is partly formed, principally through interaction of F101 and I104 in H1, which contact residues from all the other three helices; H5 also contributes significantly to core packing, via H160 and L161 at the N-terminal end. The central core residue in H2, L115, which packs onto residues in both H1 and H5 in the native state, has a medium Φ-value. Notably, although H4 is apparently well structured, the only core residue that contributes structure in the TS is V141 at the extreme N-terminus of H4. It has a high Φ-value, and appears to pin this end of H4 to H1 via an interaction with I104. In the native state, the central core of FADD DD is dominated at one end by W148F from H4, which has a Φ-value of zero. Thus, one end of the central core appears to be largely unstructured, and H4 is essentially attached only via contacts with H2 (via the B2 core)."}